Effects Of Aconitine On Connexin43 Phosphorylation Status And [Ca²⁺] Oscillation Patterns In Cultured Neonatal Rat Ventricular Myocytes

[Background]Aconitium plants are a group of important poisonous plants, belonging to the Ranunculaceae family of Angiosperm subphylum. They are one of the earliest recorded poisonous plants in China. More than 350 species of this plant have been identified in the world. They are widely distributed in the mountains or cooler regions of northern hemisphere. Up till now, over two hundred species of Aconitium plants are found in China except for Hainan Province.Aconitium plants and their preparations have been used in Chinese folk recipe and traditional medicine for more than two thousands years for their particularly curative effects. It has been confirmed by modern pharmacological studies that most of Aconitium medicinal materials have wide spread pharmaceutical properties including anti-inflammatory, detumescence, anesthetic, analgesic, cardio-tonic, hypotensive, immunosuppressant, tumor-suppressant effects and so on. Thus Aconitium plants possess considerable pharmaceutical value, however a set of various Aconitium alkaloids, belonging to the group of diesterditerpene alkaloids, are present in the tubers (root), stalks, flowers, and leaves of these plant. The substance aconitine is the most toxic Aconitium alkaloids. Aconitine poisoning is a matter of common occurrence in China, lies in the insignificant difference in aconitine dosage among therapy, poisoning and death; the individual differences in pharmaceutical tolerance of aconitine; improper or accidental take in common people; and put in poison by homicide. In resent years, suicides and homicides cases relating to aconitine poisoning are reported occasionally in Japan and some Western countries.The toxicological effects of aconitine act mainly on the nervous system and the heart. Life-threatening arrhythmias, including ventricular tachycardia and ventricular fibrillation, are the major reasons for the aconitine induced death. By using of the structure analysis of ion channels and the whole cell patch clamp technique, it had been verified that aconitine can bind with high affinity to the open state of voltage-gated sodium channels, suppress their inactivation, and facilitate the excitation of the membrane, by increasing Na⁺ in-flow and prolonging depolarization, which bring about various types of ventricular tachyarrhythmia. It should be noticed that since the 1990', accumulated result from experimental and clinical studies have shown that the electrical uncoupling among cardiomyocytes is closely related to the occurrence tachyarrhythmia. Therefore, study of the toxicology effects of aconitine on ion channels in single cardiomyocytes can not comprehensively reflect the structural and functional changes in aconitine treated cardiomyocytes which work as co-functional syncytium. On the other hand, some researchers hypothesized that theoretically, the aconitine-induced sustained influx of Na⁺ may eventually lead to intracellular Ca²⁺ overload via a Na⁺–Ca²⁺ exchange mechanism. Therefore, real-time measurement of intracellular [Ca²⁺] changes might be necessary to elucidate aconitine-induced Ca²⁺ disequilibrium in cardiomyocytes.Accompany with the development of experimental methods and possessing the advantages of simplicity, reliability, strong comparability and good reproduction, cell culture techniques have been widely apply in biological and medical realms. In vitro cultured cardiomyocytes not only maintain certain characteristics (e.g. spontaneous synchronously beating), but also eliminate the affects of complicated organism factors from neuron and humor. It is suitable for scientific research in physiology, pathology, pharmacology and toxicology at cellular and molecular level. By using of cardiomyocyte culture, we could study the toxicological effects of poisons on the structure, function and metabolism of cardiomyocytes under controlled conditions (e.g. dose, time and ways of poisonous). Therefore, the in vitro cell culture technique is worthy to be applied and popularized in forensic toxicological studies. [Objectives]1. To set up an experiment model of forensic toxicology under in vitro cell culture conditions, for incubation of the cultured neonatal rat ventricular myocytes with different dosages of standard aconitine, and investigating the aconitine induced toxicological effects on cultured cardiomyocytes;2. To exam the expressing of total amount of gap junctional protein– Connexin43 (Cx43) and the quantitative changes between phosphorylated Cx43 and non-phosphorylated Cx43 subtypes in the cultured neonatal rat ventricular myocytes before and after incubation of the cultures with aconitine;3. From the purpose of protein phosphorylation study, using functional antibodies to investigate the phosphorylation status at specific amino acid residues in the C-terminal domain of gap junctional Cx43 in cardiomyocytes at morphological level;4. Applying fluo-4 NW (no wash) fluorescent Ca²⁺ indicator, to real-timely measure the amplitude and the frequency changes of intracellular [Ca²⁺] oscillation in cardiomyocytes before and after incubated the cultures with aconitine. And then to further approach the internal relations between the changes of intracellular [Ca²⁺] oscillation pattern and the changes of Cx43 phosphorylation status in aconitine treated cultured cardiomyocytes.[Methods]1. Setting up toxicological model of neonatal rat ventricular myocytes by aconitine under in vitro culture conditionsSPF (Specific Pathogen Free) grade, 1-2 day old healthy Sprague–Dawley rats were selected, regardless of the gender. In each separate experimental batch, ten rats were used for in vitro ventricular myocytes culture. Toxicological experiments were initiated on day 6 of culture. After replenishing with serum free medium, the cultures were incubated for another 12 hours. Then the cultured cardiomyocytes were incubated with aconitine and were examined, according to the experiment design. Control groups were set each time.2. Examining the expression changes in total Cx43 protein and its phosphorylated or nonphosphorylated isoforms in cultured cardiomyocytes treated with different dosages of aconitine The cultured cardiomyocytes were incubated with aconitine at 6 different concentrations of 0.25, 0.5, 0.75, 1.0, 1.5 and 2.0μM/L. after 1 hour of incubation, total cellular membrane protein of cultured cardiomyocytes were extracted and then separated by electrophoresis. Applying the quantitative Western blot analysis, we examined the changes in total Cx43 protein and its phosphorylated or nonphosphorylated subtypes in samples of control or aconitine treated cardiomyocytes.3. Observing the changes of Cx43 phosphorylation status in its C-terminal domain at specific residues in cultured cardiomyocytes before or after aconitine incubationTwo kinds of functional antibodies: rabbit anti-Ser368 phosphorylated Cx43 and mouse anti-Ser368 nonphosphorylated Cx43 were used in our experiment. We applied laser scanning confocal microscopy and quantitative analysis for cellular fluorescent images to detect the Cx43 phosphorylation status changes in the C-terminal domain at the 368th serine amino acid residue (Ser368) in cardiomyocytes from controls or cultures treated with 1 hour of aconitine.4. Real-time dynamic detection of the changes in [Ca²⁺] oscillation patterns in cultured cardiomyocytes before or after aconitine incubationAfter removing of the serum free medium, fluo-4 NW loading solutions (blank or containing 1.00μM/L aconitine) were added to the culture wells for fluorescent indication of free Ca²⁺ in cardiomyocytes. We applied laser scanning confocal microscopy, under the rectangle scanning mode, to monitor and record the changes of [Ca²⁺] oscillation patterns in cardiomyocytes from controls or cultures treated with 1 hour of aconitine.5. Statistical analysis of experiment dataData are expressed as mean±SD. In western blot studies, differences of Cx43 isoform levels between groups were analyzed with univariate two-way analysis of variance (ANOVA); and multiple comparisons were made using Games-Howell post hoc test; differences of phosphorylated and nonphosphorylated Cx43 protein percentage between groups were determined by Kendall's W nonparametric test. In immunofluorescence detection, proportional differences of Ser368 phosphorylated or nonphosphorylated Cx43 signal positive areas were analyzed by nested ANOVA. A value of P < 0.05 was considered statistically significant. [Results]â… : Morphology, degree of purity and morphological contrast of neonatal rat ventricular myocytes from control cultures or aconitine treated cultures1. When plated in culture well, the cardiomyocytes were bright pellets in shape and suspended in medium. After 12 hours, they began to crawl on the bottom of the culture well and spontaneous beating could be found in few cells. After 24 hours, attached myocytes connected each other, looked like a net; and slow synchronous beating could be found in all cultures. After 48 hours, the cardiomyocytes stretched on the bottles, and high-speed (120 times per minute) synchronous beating monolayers of syncytium were formed.2. On day 6 of culture, we applied immunofluorescent microscopy and identified the cultures, using mouse monoclonal anti cTnI antibody. We estimated that > 95% of the cells were cardiac myocytes.3. It was verified by H.E. staining that compared to the control cultures, the morphological structure of cardiomyocytes were hardly changed after incubation of the cultures with different dosages of aconitine.â…¡: The effects of different dosages of aconitine on the amount of total Cx43 protein and its phosphorylated or nonphosphorylated subtypes1. Three Cx43 protein bands: 43 kDa, 40 kDa and 39 kDa in SDS-polyacrylamide gel could be detected by rabbit anti-total Cx43 antibody. These bands represent two phosphorylated isoforms (P2 and P1) and a non-phosphorylated isoform (NP) respectively. Under control conditions, most of Cx43 accumulate at 43 kDa (P2 band); after treatment of the cultures with alkaline phosphatase, the overall band migrates forward to 39 kDa (NP band);2. After 0.25μM/L aconitine treatment, the band of Cx43 migrated forward, and the P1 band could be detected; after 0.5μM/L aconitine treatment, the NP band emerged; when the concentration of aconitine was elevated to 1.0μM/L, the intensity of NP band increased significantly, concomitant with significant decreased intensity of band P2 and band P1; the effects of 1.5 or 2.0μM/L aconitine were about equal to that of 1.0μM/L aconitine;3. Quantitative analysis of Cx43 band intensity showed that there were no significant differences in total amount of Cx43 in cardiomyocytes among controls, alkaline phosphatase treated cultures or aconitine treated cultures; however, after aconitine treatment, the phosphorylated Cx43 (P2 + P1) decreased, concomitant with the increase in nonphosphorylated Cx43. The observations indicated that treatment of cardiomyocytes with aconitine did not affect the total amount of Cx43, whereas aconitine induced the dephosphorylation of Cx43 and present a concentration-dependent effect.â…¢: The effects of aconitine on the phosphorylation status in Cx43 C-terminal domain at specific residues1. In the present study, we examined the Cx43 phosphorylation status changes in the C-terminal domain at the 368th serine amino acid residue (Ser368). Results from immunofluorescent staining revealed that under control conditions, P-Cx43 (Ser368) positive gap junction plaques (green fluorescence) were sparse as broken lines along the perimeters of cardiomyocytes; in contrast, only a few particles of NP-Cx43 (Ser368) positive signal (red fluorescence) were seen in the cytoplasm;2. Incubation of the cultures with aconitine led to a dramatic loss of P-Cx43 (Ser368); whereas the NP-Cx43 (Ser368) particles were found abundantly accumulated at the points of intercellular borders. Quantitative immunofluorescent data indicate that compared to the controls, treatment of the cultures with aconitine resulted in a distinct decrease in P-Cx43 (Ser368) concomitant with a distinct increase in NP-Cx43 (Ser368). So, it is clear that aconitine-induced dephosphorylation of Cx43 occurs at Ser368 in its C-terminal domain.â…£: The effects of aconitine on the intracellular [Ca²⁺] oscillation patterns in cultured cardiomyocytes1. No matter whether treated with aconitine or not, the [Ca²⁺] oscillated synchronously in both nuclear and cytoplasmic regions of a same cardiomyocyte;2. Under control conditions, [Ca²⁺] oscillations were irregular but relatively stable accompanied by occasional small calcium sparks;3. After incubation of the cultures with aconitine, strenuous high frequency [Ca²⁺] oscillations emerged in both nuclear and cytoplasmic regions and typical calcium sparks disappeared; however, the average [Ca²⁺] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. [Conclusions]1. In the selected concentration range (0.25 2.0μM/L), different dosages of aconitine do not disrupt the conformational structure of cultured cardiomyocytes;2. Compared to the controls, after treatment of the cardiomyocytes with different dosages of aconitine, the total amounts of Cx43 do not change, which indicate that aconitine do not interfere the expression of Cx43 protein;3. Accompanied by the elevation of aconitine concentration, the phosphorylated Cx43 (P2 + P1) decreased, concomitant with an increase in the nonphosphorylated Cx43. The observations indicated that in cultured cardiomyocytes, aconitine induced the dephosphorylation of Cx43 and present a concentration-dependent effect.4. Under the control conditions, the 368th serine amino acid residue (Ser368), which is located in the C-terminal domain of Cx43, is phosphorylated. After treatment of the cultures with aconitine, the Ser368 residue undergoes dephosphorylation. Since the phosphorylation status of Cx43 is of very importance to the maintenance of the electric coupling among cardiomyocytes, inducing the dephosphorylation of Cx43 at specific residue underlies the mechanisms of aconitine intoxication in cardiomyocytes.5. Aconitine can bind with high affinity to the open state of voltage-gated sodium channels; suppress their inactivation and increase Na⁺ in-flow. Theoretically, the aconitine-induced sustained influx of Na⁺ may eventually lead to intracellular Ca²⁺ overload via a Na⁺–Ca²⁺ exchange mechanism. In the present study, we found that although oscillated strenuously at high frequency, the average [Ca²⁺] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. The observations indicate that aconitine induce the changes in [Ca²⁺] oscillation frequency other than the Ca²⁺ overload in cardiomyocytes, which may affects some [Ca²⁺] oscillation frequency sensitive process of signal transduction in cardiomyocytes.